Project Summary/Abstract The mammalian heart forms early in embryogenesis, and defects in its formation are at the root of congenital heart defects (CHDs), affecting 1?2% of live births. In adulthood, heart disease is the number one killer in the Western world, resulting in a considerable health burden. The terminally differentiation state of the postnatal heart means that damaged myocardium is permanently lost upon injury (e.g., after a myocardial infarction). Understanding the building blocks of the heart has clear importance not only for defining the etiology of CHDs, but also for creating novel cell-based regeneration strategies to treat heart disease. The origins of the heart in embryogenesis have been defined as beginning in mesoderm that arises during gastrulation. We have identified Smarcd3 as marking an early subpopulation of Mesp1 lineage-labeled cells, which contribute almost exclusively to the heart. We have already identified subpopulations within this progenitor pool, which contribute to specific anatomical structures of the forming heart. Specifically, Tbx5 lineage-labeled cells contribute to the atria and left ventricle, while the Mef2AHF lineage labeled cells contribute largely to the outflow tract and right ventricle. Using a novel dual-lineage labeling method, we determined that in addition to these two broad populations, there is a third subset of cells labeled by both markers in the early embryo that contribute exquisitely and selectively to one side of the interventricular septum, forming a sharp lineage boundary. This surprising finding suggests a very early and refined patterning of cardiogenic mesoderm, with some progenitors already destined to a future anatomical location, ahead of morphogenesis. We have determined that reduced TBX5 dosage, which results in ventricular septation defects, disrupts the integrity of the lineage boundary. This for the first time allows a molecular genetic dissection of the mechanisms regulating a specific population of cells that are essential for septal formation. These findings redefine the origins of the cardiac chambers, and provide exciting new avenues to understand cardiac cell fate and morphogenesis. In this proposal using transgenic reporter lines combined with single cell RNA-seq we will in an unbiased manner define the range of early cardiac progenitors that populate the late mouse gastrula. We will also define the contribution of specific genetically labeled populations by clonal lineage tracing. We will define the origins, identity, and migration of a specific cell population that contributes exclusively to the interventricular septum. We will examine the effect of disrupting the boundary that this lineage forms, by reduced dosage of its regulator, TBX5, and by deleting this cell population prior to or during ventricular septal morphogenesis. Finally, We will determine the effects of eliminating the function of specific transcriptional regulators of mesoderm and cardiac differentiation on the establishment and migration of early cardiac lineages.